The invention relates to a method for preventing chemical crosstalk in enzyme-linked reactions using a microreaction array having at least two reaction chambers for receiving substances which react chemically or biochemically with other substances. In addition, the invention also relates to a system for carrying out the method.
Combinatorial analysis and synthesis are nowadays in increasingly widespread use for the development of new active ingredients in the life sciences industry (pharmaceuticals), food technology, agro technology (crop science), in medical diagnostics and also to solve a very wide range of objectives in general biotechnology. To carry out these methods, what are known as microtitration plate techniques with reaction wells in an array structure are used, employing either 96 or even 384 wells for simultaneous reaction on an array surface of, for example, approx. 12×8 cm2. The density of these arrays will increase further in future, which means that different types of chemical reactions have to take place in reaction chambers arranged ever closer together.
U.S. Pat. No. 6,143,496 A has disclosed a PCR (Polymerase Chain Reaction) method, in which, in an array having a multiplicity of reaction chambers, individual reactions take place next to one another at elevated temperatures. In this array, there are suitable ways to isolate specimen chambers, which can also be achieved, for example, by a displacement liquid. In particular, it is important to reduce the specimen volume or to prevent evaporation of water. The same problem in connection with a PCR method is discussed in WO 01/34842 A2, which claims an earlier priority but was not published before the priority date of the present application.
The situation is in principle different with what are known as DNA chips, as are known from various publications. The situation is taken to extremes, for example, with an array of different DNA probe molecules which are arranged at a spacing of only a few tens of micrometers and with a density of, for example, a few hundred positions per few mm2 on a planar substrate, known as the DNA chip. If molecules which can move freely are involved in the analytical detection of, for example, unknown DNA, chemical crosstalk occurs with such dense arrays.
For a plurality of reasons, for example on account of the high specificity and the low detection limit, biochemical analysis often uses enzyme-linked detection methods. By way of example, what are known as ELISA (Enzyme-Linked ImmunoSorbent Assay) tests are in widespread use in medical diagnostics and in the research sector. (Literature reference c.f. B. Alberts et al. (eds.), Molekularbiologie der Zelle (Molecular biology of the Cell) (1997), 3rd edition, page 216, VCH Weinheim). Methods using enzyme markers in a known redox (re)cycling are also employed for applications in the field of the DNA chip (A.v.d.Berg, P. Bergveld (eds.) Proceedings of the μTAS '94 Workshop (1994), pp. 249 to 254, Kluwer Academic Publishers Dordrecht).
In all cases mentioned in the specialist literature, the enzyme is not free in the liquid phase of the arrangement, which is also known as an assay, but rather is bonded and therefore, as an “enzyme label” marks the primary substance to be detected. In this case, the bonding of the enzyme molecules to the substance to be detected is always stoichiometric. Amplification occurs in that the enzyme converts added substrate molecules at high speed. This conversion is quantified, for example, optically or electrochemically, depending on the substrate used and/or the product formed. For this purpose, irrespective of the method used, in particular the increase in concentration of the product P, i.e. the time-dependent function dc(P)/dt, is monitored.
If assays of this type are carried out in an array, as described in detail in the related art, reaction products which can move freely and are formed by the enzyme can also reach adjacent enzyme-free array positions, where they may simulate the presence of the enzyme label. This phenomenon is known as crosstalk, which leads to measurement errors and may therefore give false results.